Protein synthesis is one of the central cellular processes. We investigate eukaryotic translation by reconstituting in vitro from individual mammalian components: ribosomal subunits, 14 translation factors, aminoacylated tRNAs, and mRNAs. Translation initiation on mRNAs with highly structured 5'-untranslated regions (5'UTRs), which encode the majority of cellular proteins, strongly requires DExH-box helicase DHX29. DHX29 plays the physiologically important role in cell growth and tumorigenesis. One objective of this proposal is to establish structure-function relations of DHX29 and to determine the mechanism of DHX29-mediated translation initiation on mRNAs with highly structured 5'UTRs. Mutational analysis will be employed to assign the role of DHX29's motifs/domains in the association with 40S subunit and initiation factors as well as in mediating of initiation. Since DHX29 alone is not a processive RNA helicase and some DExH-box helicases require cofactor, potential DHX29's cofactor will be searched by means of the cellular lysate fractionation. Also DHX29 will be substituted for its poorly studied sequence paralogs (DHXs 9, 16, 30, 38, and 57) in our system for the following comparative protein analysis. The effect of DHX29 on IRES-driven translation initiation of viral mRNAs, which is the alternative mechanism to canonical 5'-end dependent translation initiation, will be studied at the cellular level. Cryo-electron microscopy approach will be employed for the comprehensive structural characterization of DHX29-associated ribosomal complexes. The oncogenic mRNAs have the characteristic highly structured 5'UTRs and their translation is elevated in cancer cells, therefore, detailed understanding of the mechanism of DHX29-mediated translation initiation will facilitate the search of ways for the targeted translation regulation of these mRNAs. No-Go Decay (NGD) and Non-Stop Decay (NSD) are essential cellular mRNA quality control pathways in eukaryotes aiming to detect stalled translation elongation ribosomal complexes, to dissociate them and to destroy defective mRNAs. Stalled NGD/NSD complexes in mammals are efficiently dissociated by Pelota/Hbs1/ABCE1 and the mRNA degradation of stalled NGD complexes in HeLa lysate is initiated by the endonucleolytic cleavage in the ribosomal A-site, but the endonuclease is unknown. Therefore, to get insights into NGD/NSD, another objective of this proposal is to determine structure-function relations of human Pelota in the dissociation of stalled complexes and to identify the endonuclease. Mutagenesis of recombinant Pelota will be used to address the role of its motifs/domains in the association with 80S ribosomes and ABCE1 as well as in mediating of the dissociation process. To identify the endonuclease, HeLa lysate fractionation approach combined with the developed functional test for the mRNA cleavage observation in reconstituted stalled complexes will be utilized. Several genetic deceases are associated with downregulated expression of mRNAs which are targets for NSD, and the obtained data will create the prerequisites for the therapy development to control the expression of disease-associated mRNAs. PUBLIC HEALTH RELEVANCE: On the one hand, DExH-box helicase DHX29 is important for eukaryotic translation initiation on mRNAs with highly structured 5'UTRs and plays the physiologically important role in cell growth and tumorigenesis, and, on the other hand, the oncogenic mRNAs have the characteristic highly structured 5'UTRs and their translation is elevated in cancer cells, therefore, translational inhibition through DHX29 silencing might present a means by which cancer cell growth could be inhibited and detailed understanding of the mechanism of DHX29-mediated translation initiation will facilitate the search of the targeted DHX29 silencing. Several genetic deceases (e.g. factor FX mediated inherited bleeding disorders, WAS protein associated Wiskott-Aldrich syndrome, and S19 ribosomal protein linked Diamond-Blackfan anemia) are shown to be associated with downregulated expression of mRNAs which are targets for NSD, and, thus, the obtained insights into NSD process will create the prerequisites for the therapy development to control the expression of disease-associated mRNAs.